HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

Summary

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

Abstract

The heteromeric receptor complex between 5-HT_2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis^1,2. Consequently, investigation of structural details of the interaction between 5-HT_2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT_2A knockout (KO) mice^3,4. Additionally, by conditionally expressing the 5-HT_2A receptor only in cortex, it was demonstrated that 5-HT_2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs³. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice⁵. These findings suggest that mGlu2 is at least in part necessary for the 5-HT_2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT_2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2ΔTM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT_2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs⁶.

Introduction

Hallucinogens, such as LSD, psilocybin and mescaline cause significant changes in human consciousness, cognition and emotion^7-9. Inactivation of serotonin 5-HT_2A receptor signaling by either genetic or pharmacological approaches causes markedly attenuated behavioral responses to hallucinogens in both rodent models^3,10 and humans¹¹. Although hallucinogens bind other receptor subtypes⁸, the 5-HT_2A receptor is considered as necessary for the unique behavioral activity of these chemicals.

Group II metabotropic glutamate receptors (i.e., mGlu2 and mGlu3) have been the tar....

Protocol

NOTE: All procedures for animal breeding and cares were conducted according to the Institutional Animal Care and Use Committee (IACUC) regulation of Icahn School of Medicine at Mount Sinai. Be sure to use sterile gloves throughout the procedure. 

1. Drug and Virus Preparation

1. Drug Preparation
   1. Prepare 15.0 ml ketamine/xylazine anesthetic by dissolving 1.35 ml of 100 mg/ml Ketamine and 0.75 ml of 20 mg/ml xylazine in 12.9 ml of 0.9% Saline solution. Thoroughly mix solution.
2. Virus Preparation
   1. Clone the mGlu2 and mGlu2ΔTM4N constructs into a bicistronic herp....

Results

Previous findings demonstrate that the head-twitch murine behavioral response is reliably and robustly elicited by hallucinogens, and it is absent in 5-HT_2A-KO mice³. Furthermore, it has been shown that the head-twitch response elicited by the hallucinogenic 5-HT_2A agonists DOI and LSD was significantly decreased in mGlu2-KO mice⁵. However, although previous findings convincingly demonstrate that 5-HT_2A and mGlu2 are assembled as a he.......

Discussion

Together with previous findings in mGlu2-KO mice⁵, the results with mGlu2 and mGlu2/mGlu3 chimeric constructs that do not form the 5-HT_2A-mGlu2 receptor complex in cultured cells suggest that the 5-HT_2A -mGlu2 heteromeric receptor complex in mouse frontal cortex is needed to induce head-twitch behavior by LSD-like hallucinogenic 5-HT_2A receptor agonists. A limitation of this method is that it does not measure close molecular proximity at a subcellular level in native tissue. In.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
mGlu2 bicitronic herpes simplex virus (HSV) vector	MIT Core		mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)
mGlu2ΔTM4N bicitronic herpes simplex virus (HSV) vector	MIT Core		mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)
GFP bicitronic herpes simplex virus (HSV) vector	MIT Core		mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)
Xylazine	Lloyd	List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10	1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution
Ketamine	Vedco	KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06	1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution
Ophthalmic gel	Fisher Scientific	NC0550805
Burret clips	Fisher Scientific	NC9268369
Feather surgical blade	Fisher Scientific	NC9032736
Hydrogen Peroxide	Fisher Scientific	19-898-919
Hamilton syringe	Fisher Scientific	14815203
Hamilton™ Small Hub Removable Needles (33 Ga)	Fisher Scientific	14816206
Cordless Micro Drill	Fisher Scientific	NC9089241
Dermabond Dermal Adhesive	Fisher Scientific	NC0690470
(±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)	Sigma-Aldrich	42203-78-1	Dissolved in 0.9% saline solution to the concentration of 2.0 mg/kg